流式细胞仪荧光补偿

流式细胞仪荧光补偿 流式细胞仪荧光补偿(一) 在流式细胞仪分析中用到的“荧光补偿”这个术语是指修正荧光渗漏的过程,如从探测器除去除匹配荧光以外的任何荧光信号。荧光补偿过程从原理上来说相对简单，但实际上涉及许多精细、敏感的方面从而使在实践操作相当复杂。 非常不幸，荧光补偿操作可能是在流式操作中的数据采集和分析等步骤中最少被了解和掌握的，可能是因为在介绍它时经常要用到线性代数来计算，并且还要求了解它的产生原理。然而，正确的荧光补偿对于许多流式分析来说绝对是至关重要的，特别是对现常见的多抗原荧光达强度分析。因为荧光补偿常被误解、误用并被许多不正确的观念所包围，许多实验室的荧光补偿设置都不正确。本章的一个部分将剖析有关补偿的一些疑惑问题。 图1显示在设置荧光补偿中常犯错误。本章将致力于从以下方面阐述流式细胞仪荧光补偿问题:为什么它是必须的，它是如何通过硬件或软件来完成的，荧光补偿是如何时影响数据视觉效果的，最后是如何设计一个最佳的实验来获得一个正确的荧光补偿。通过完整地阅读本章节，读者将学会为实验正确地设置荧光补偿并在出版物中识别出补偿设置不当的数据，并能够明白这些错误是如何影响分析结果的。尽管全面的阐述荧光补偿不可避免地要涉及到一些线性代数的问题，但正确掌握荧光补偿并不一定要求要掌握它;在这两部分内容中的问题可根据你的意愿撇去或跳过。 大部分的流式用户不能正确回答图1中的三个问题。对于问题1的正确答案是“2”。对于问题2，答案中十字门虚线的位置就不正确。最后，第三个问题的正确答案是:“不可能决定哪个图形中的荧光补偿是正确的”，因此其中没有哪个可以说中正确的。 如果一个正确的荧光补偿对于流式数据分析这么重要，并且又这么少的人知道如何正确地去设置它，而为什么到目前为止流式细胞仪的分析结果还都不错。对于此问题的简单答案是绝大多数类型的数据分析并不绝对需要正确的荧光补偿。此问题的另一个答案是不正确的荧光补偿在双色或三色分析中显得并不十分重要。然而随着五色分在大多数实验室中开展，如果这些个问题持续下去将会导致实验失败。 举例来说，思考在图1中的图谱，可以发现即使荧光补偿并不正确但仍可以正确计算细胞亚群的比例。然而计算这些细胞亚群的比例必须以单染的样本为对照，而不是以各个荧光的同型对照染色为对照～正确的设门应该是以CD3FITC和PEisotype染色样本为基础。 图1中还显示了荧光补偿在正确检测抗原荧光表达强度中重要影响。在此，任何不正确的荧光渗漏都会误导分析结果。此外，正确的荧光补偿在将弱阳性群体与阴性群体正确区分中有极其重要的作用;荧光补偿不足会导致弱阳性群体是假阳性颗粒增多，而过度补偿则会丢失弱阳性群体而导致假阴性。 注意在补偿调节不当的数据中正确计算的亚群数量的现象在三色或更多荧光分析实验中可能不成立。在本章的结尾将给出一个计算失败的这种案例。此外还讨论了如何为分析数据设计一个正确的染色对照(甚至当荧光补偿设置并不正确时)。这些称为“缺一型荧光染色”或FMO对照是进行正确多色荧光分析的前提保证。 荧光补偿基础 荧光补偿是指修正荧光光学信号在探测器之间相互渗漏并被数字化检测的过程。 (3) construct safe volunteers is realistic need to innovate the social management style. Only continuous innovation, our management to adapt to the changing social needs. Especially in today's social security situation is complex, criminal activity appeared subtle, flow, information, science and technology, gangs of new features only continuous innovation in order preventive measures, we can in an invincible position and fight criminals. We set up the peace volunteers, both volunteers is ordinary people, always living in the crowd, for the first time found the problem, information most really the most solid, it is a kind of innovation of the social management is A positive exploration of the mode of social security. Two, strengthen the sense of innovation, do a good job in combination with the article, to promote the smooth development of the public security work One is to with the Bureau of the county Party committee work combined. To carefully control the "Four Construction Bureau of county Party committee deployment project decomposition and the division of tasks, and to identify volunteers safe construction of the starting point, combined with point and the breakthrough point, to achieve the same resonance frequency, benign interaction. Que Baozhi may work and the public security work achieved double harvest. 图1 你能看出以上哪个样本的补偿是正确的吗,在这个假设实验中模拟了外周血用FITC标记的CD3和PE标记 的阴性对照作为质控物。在(A)和(B)中，居左上方的图形显示了未调节补偿的数据。每张以数字编号的图 形显示补偿设置逐渐增加的过程(FITC与PE两探测器之间)。在B组图中，显示了同一样本的检测数据，但在 信号收集时PE探测器的电压很低。请回答问题:(1)在A组图中(1、2、3、4)哪个补偿调节是正确的,(2) 你是依据什么来判断的,(3)在B组图中哪个补偿调节是正确的,请在本文的介绍部分参阅以上问题的答案。 (3) construct safe volunteers is realistic need to innovate the social management style. Only continuous innovation, our management to adapt to the changing social needs. Especially in today's social security situation is complex, criminal activity appeared subtle, flow, information, science and technology, gangs of new features only continuous innovation in order preventive measures, we can in an invincible position and fight criminals. We set up the peace volunteers, both volunteers is ordinary people, always living in the crowd, for the first time found the problem, information most really the most solid, it is a kind of innovation of the social management is A positive exploration of the mode of social security. Two, strengthen the sense of innovation, do a good job in combination with the article, to promote the smooth development of the public security work One is to with the Bureau of the county Party committee work combined. To carefully control the "Four Construction Bureau of county Party committee deployment project decomposition and the division of tasks, and to identify volunteers safe construction of the starting point, combined with point and the breakthrough point, to achieve the same resonance frequency, benign interaction. Que Baozhi may work and the public security work achieved double harvest. 自单激光双色分析出现后此过程变得十分重要。每种荧光素分子都具有自身的光谱发射范围。这些发射光谱之间存在相互叠加，在某些情况下此现象十分明显。举例来说，如图2为FTIC与PE荧光发射光谱的叠加情况。在一个双探测器系统中，可设计将FITC荧光从PE荧光中分离出来。检测荧光发射光时根据其波长和分布将选择其中一小部分进入探测器，这部分荧光可由不同的滤光片从全部发射光中选择特定波长范围的荧光来获得。这样，FTIC荧由530nm的滤光片选择后送入探测器中;PE荧光则是由575nm滤光片来选择。但是有部分FITC荧光会出现在PE探测器中，这是因为它的发射光涵盖两个探测器滤片的过滤范围。这部分信号我们称之为荧光渗漏，因为它是由FITC荧光渗漏到PE探测器中的。注意想要设计一组荧光滤片来只收集FITC或PE信号是不可能的;因为当同事使用这两种荧光染料时荧光叠加总是存在的。这样只要FITC荧光存在，就会在530nm区得到一个信号，同时也会在575nm区得另一些信号。如这时有PE荧光存在，它也将在575nm区收集。那我们如何才能计数多少信号是来自PE而又有多少信号是由FITC引起的呢,这个计算过程我们称为“荧光补偿”，如去除FITC在PE发射光谱带上的荧光来修正PE探测器中的信号。 一步法荧光补偿 如果一种荧光染料的发射光可以被两个含有不同波段的滤光片的探测器检测到(如，在发射光谱的不同区域检测)，那么就能根据其中一个探测器的信号计算出另一个探测中有多少信号。这是因为这两个信号是成比例变化的(如图3)。这个恒定特性是指我们能够根据绿色荧光探测器中的曲线面积(绿色荧光信号)来精确推算出橙色荧光探测器的曲线面积(如:橙色荧光信号)。 这两个值的比值可在没有FITC以外荧光的情况下计算得知。用于此目的的样本被称为“荧光补偿调节样本”或是荧光补偿质控物，用于精确测定补偿值。对于典型的流式细胞仪来说，FITC发射光在橙色探测器中的信号大概是绿色探测器的15%。因此，如果我们从橙色探测器中减秒绿色探测器信号的15%，那无论有多少FITC信号存在，经校准后的橙色荧光探测器信号将总为零。这时可在系统中加入PE荧光，橙色荧光探测器经扣除了15%的绿色信号后将显示真正的PE荧光信号，而不用再考虑是否存在FTIC荧光了。这就是“一步法”荧光补偿:在第二探测器中修正来自另一荧光的信号。 二步法(双)荧光补偿 从图3的荧光谱中可见PE荧光也有部分渗漏至FITC探测器中。通过检测只染有PE荧光的细胞来获知此比例，一般它的数值是2%左右。这称之为PE荧光补偿校正物。 但如果PE荧光出现在FITC探测器中，而同时我们又使用FITC荧光来修正PE探测器中的荧光信号，那这还能有可能来正确修正荧光渗漏来得真正的荧光信号吗,一个不完整的答案来解释此问题:在现有n色荧光分析实验，n是指在仪器上同时检测不确定的荧光数量;这个系统的荧光补偿功能可以完全解决此问题。实际上，根据一些数学运算是有可能使用这种方法来精确调节补偿的(读者可以跳过下一章节，而不必研究其中原理)。 xF定义为在n探测器中检测到的来自x荧光的信号。这样fF1就是指FITC探测器中绿色荧光的信n 号;pF2是指橙色荧光探测器是PE荧光的信号。D是指探测n所检测到的荧光信号。在此情况n 下，假设没有任可背景荧光信号;背景荧光信号将稍后讨论。于是: 1 (3) construct safe volunteers is realistic need to innovate the social management style. Only continuous innovation, our management to adapt to the changing social needs. Especially in today's social security situation is complex, criminal activity appeared subtle, flow, information, science and technology, gangs of new features only continuous innovation in order preventive measures, we can in an invincible position and fight criminals. We set up the peace volunteers, both volunteers is ordinary people, always living in the crowd, for the first time found the problem, information most really the most solid, it is a kind of innovation of the social management is A positive exploration of the mode of social security. Two, strengthen the sense of innovation, do a good job in combination with the article, to promote the smooth development of the public security work One is to with the Bureau of the county Party committee work combined. To carefully control the "Four Construction Bureau of county Party committee deployment project decomposition and the division of tasks, and to identify volunteers safe construction of the starting point, combined with point and the breakthrough point, to achieve the same resonance frequency, benign interaction. Que Baozhi may work and the public security work achieved double harvest. 图2 (3) construct safe volunteers is realistic need to innovate the social management style. Only continuous innovation, our management to adapt to the changing social needs. Especially in today's social security situation is complex, criminal activity appeared subtle, flow, information, science and technology, gangs of new features only continuous innovation in order preventive measures, we can in an invincible position and fight criminals. We set up the peace volunteers, both volunteers is ordinary people, always living in the crowd, for the first time found the problem, information most really the most solid, it is a kind of innovation of the social management is A positive exploration of the mode of social security. Two, strengthen the sense of innovation, do a good job in combination with the article, to promote the smooth development of the public security work One is to with the Bureau of the county Party committee work combined. To carefully control the "Four Construction Bureau of county Party committee deployment project decomposition and the division of tasks, and to identify volunteers safe construction of the starting point, combined with point and the breakthrough point, to achieve the same resonance frequency, benign interaction. Que Baozhi may work and the public security work achieved double harvest. 图3 FITC和PE荧光发射光光谱。发射光(由荧光分受激发后发出的光线)是在488nm光源下激发产生的(氩离子激光器发射光)。FITC发射光的峰值大约在515nm，典型的用于收集它的发射光的滤片是在530nm(深灰色区域)。PE的发射光是远红光线，峰值在575nm左右，检测它的滤片位此峰值中央。注意FITC的发射光有部分延伸至PE信号收集范围(B);通常落在575nm波段的荧光信号约为530nm波段荧光信号的15%。PE的发射光只有极少部分落在530nm波段中(C)，通常它只占575nm波段信号的2%(D)。 图2 ( 左侧) 一个补偿调节不正确的数据仍能被分析。 这些图谱数据是外周血经CD3FITC和CD4PE或PE同型对照染色检测的结果。顶端的图显示的是未经补偿调节的数据;列图中显示了FITC到PE中的补偿依次增加后的结果。十字虚线是依据未染色样本(或是经两种荧光的同型对照染色)设定的来定义阳性细胞群体。实线门是依据单荧光-同型对照染色设定的。注意在任何一张图中，不用考虑补偿是否正确，都可正确计算CD3阳性细胞中表达CD4(CD3+CD4+)细胞的数据，但必须是基于单荧光-同型对照染色对照上的(左侧)。完全以同型对照来设定的虚线十字门来统计将会导致错误的结果。仅在补偿调节正确的情况下(中间图)才能够在CD3+CD4+细胞中准确计算CD4荧光;补偿不足时CD4荧光过强而当补偿过度时出现了阴性群体。所以抗原浓度的检测对不恰当的补偿设定将会特别敏感。 (3) construct safe volunteers is realistic need to innovate the social management style. Only continuous innovation, our management to adapt to the changing social needs. Especially in today's social security situation is complex, criminal activity appeared subtle, flow, information, science and technology, gangs of new features only continuous innovation in order preventive measures, we can in an invincible position and fight criminals. We set up the peace volunteers, both volunteers is ordinary people, always living in the crowd, for the first time found the problem, information most really the most solid, it is a kind of innovation of the social management is A positive exploration of the mode of social security. Two, strengthen the sense of innovation, do a good job in combination with the article, to promote the smooth development of the public security work One is to with the Bureau of the county Party committee work combined. To carefully control the "Four Construction Bureau of county Party committee deployment project decomposition and the division of tasks, and to identify volunteers safe construction of the starting point, combined with point and the breakthrough point, to achieve the same resonance frequency, benign interaction. Que Baozhi may work and the public security work achieved double harvest. 公式2 x荧光在其他探测中的信号将与xFn计算例(n指代专门收集此荧光的探测器);这样，如要知道有多少真正的FITC或PE信号,就需要计算fF1和pF2。现已知(自图3)fF1与 fF2的比值是衡定的，就如同pF2与 pF1一样，这个衡定比值定义如下: 公式3 公式4 因此，xSn是荧光素x在其探测器荧光信号与探测器n的渗漏系数。这个值由检测补偿质控物来获得，此时样本所有荧光只来自于一个荧光素。举例来说，FITC荧光补偿质控物: 公式5 公式6 (3) construct safe volunteers is realistic need to innovate the social management style. Only continuous innovation, our management to adapt to the changing social needs. Especially in today's social security situation is complex, criminal activity appeared subtle, flow, information, science and technology, gangs of new features only continuous innovation in order preventive measures, we can in an invincible position and fight criminals. We set up the peace volunteers, both volunteers is ordinary people, always living in the crowd, for the first time found the problem, information most really the most solid, it is a kind of innovation of the social management is A positive exploration of the mode of social security. Two, strengthen the sense of innovation, do a good job in combination with the article, to promote the smooth development of the public security work One is to with the Bureau of the county Party committee work combined. To carefully control the "Four Construction Bureau of county Party committee deployment project decomposition and the division of tasks, and to identify volunteers safe construction of the starting point, combined with point and the breakthrough point, to achieve the same resonance frequency, benign interaction. Que Baozhi may work and the public security work achieved double harvest. 公式7 这样，FITC荧光渗漏系数就是FITC染色样本在各个探测器中的荧光信号与FITC探测器荧光信号的比值。类似的，PE荧光补偿质控物计算如下: 公式8 注意在公式8中D1和D2与在公式7的值是不同的，因为公式8中是指PE荧光的检测值。 一旦渗漏系统已知，它们便可用于染色样本来计算真正的荧光信号xFn。回想上述公式5、6: 公式9 公式10 在这两个公式中还有两个未知数(fF1和 pF2)，它们可被解析如下: 公式11 公式12 fF1和 pF2分别是指FITC在绿色荧光探测中的信号，PE在橙色荧光探测中的信号，这正是我们所期望的真正信号，从它的计算公式来看所有数值都是不变量，所以就不存在逻辑无限循环的情况。 (3) construct safe volunteers is realistic need to innovate the social management style. Only continuous innovation, our management to adapt to the changing social needs. Especially in today's social security situation is complex, criminal activity appeared subtle, flow, information, science and technology, gangs of new features only continuous innovation in order preventive measures, we can in an invincible position and fight criminals. We set up the peace volunteers, both volunteers is ordinary people, always living in the crowd, for the first time found the problem, information most really the most solid, it is a kind of innovation of the social management is A positive exploration of the mode of social security. Two, strengthen the sense of innovation, do a good job in combination with the article, to promote the smooth development of the public security work One is to with the Bureau of the county Party committee work combined. To carefully control the "Four Construction Bureau of county Party committee deployment project decomposition and the division of tasks, and to identify volunteers safe construction of the starting point, combined with point and the breakthrough point, to achieve the same resonance frequency, benign interaction. Que Baozhi may work and the public security work achieved double harvest.